| Background and ObjectiveNon small cell lung cancer (NSCLC) is one of the leading causes of cancer-related morbidity and mortality. Short interfering RNA (siRNA) targeting angiogenic factors, and further inhibiting tumor angiogenesis, is one of the excellent candidates for lung cancer treatment. However, the target for this strategy was quite limited, the time to recurrence was frequently short, and antiangiogenic drugs must be used in combination with traditional chemotherapy. RNA with a triphosphate group at the5’ end is the specific ligand of retinoic acid inducible gene-I (RIG-I), which was known as one of pattern recognition receptors in cytoplasm.5’-triphosphate RNA was naturally generated by RNA virus intracellular, and could activate the innate immune system. In this study, we combined the RIG-I ligand activity with siRNA targeting vascular endothelial growth factor (VEGF), and direct VEGF-specific siRNA with5’-triphosphate ends (ppp-VEGF). This kind of siRNA with5’-triphosphate ends could simulate RNA virus, and has three anti-cancer effects at the same time:1. Specific silence of VEGF, and inhibit tumor angiogenesis;2. Activate RIG-I signaling pathway, induce anti-tumor immunity, blocking the immune evasion of cancer;3. Induce endogenous apoptosis and incomplete autophagy in cancer cells, and promoted programmed cell death. ppp-VEGF might break through the limitations of traditional anti-angiogenic drugs, it has multiple target, and could be used without chemotherapy. Materials and Methods1. We developed siRNA targeting VEGF with a5’-triphosphate end (ppp-VEGF). Three lung adenocarcinoma cell lines, A549, H1299, LLC were used in this study. We transfected the cells with four kinds of siRNA:OH-Con, OH-VEGF, ppp-Con, ppp-VEGF, and used qRT-PCR, western blot, and Elisa to detect the VEGF expression at both mRNA and protein level. Try to clarify VEGF silence effects of ppp-VEGF. Furthermore, we used scratch assay to detect the effects of ppp-VEGF on the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Matrigel was adopted to figure out whether ppp-VEGF could inhibit the vascular network formation.2. OH-Con, OH-VEGF, ppp-Con, ppp-VEGF were respectively transfected into lung cancer cells, we detected the expression of inflammatory factors (IP-10, IFN-β) both intracellular and in cell supernatant. Meanwhile, we used flow cytometry to measure the percentage of MHC-I/HLA positive cells. Try to clarify whether ppp-VEGF could activate the innate immune system, and inhibit immune evasion of cancer cells. In order to explain the exact mechanism of ppp-siRNA for immune activation, we first chose western blot to detect the state of RIG-I signaling pathway. Then we developed siRNA targeting RIG-I and mitochondrial antiviral signaling (MAVS), to specific silence the expression of the above two genes, then detect the changes of ppp-siRNA activated immunity. In this way, we could express the meanings of RIG-I signaling pathway in the ppp-siRNA activated immunity.3. Annexin V/PI was chosen to quantitative the apoptotic status of cancer cells, MTT and trypan blue were used to confirm how ppp-VEGF will affect the proliferation and survival of cancer cells. How does ppp-VEGF achieved the purpose for cancer killing? To answer such questions, we first detect the expression of Noxa, which is one of the members from BH3-only family, and might induce endogenous apoptotic pathway. To be followed, as ppp-siRNA was RNA virus simulator, it might cause autophagic cell death. We measured the ratio of LC3-II/LC3-I by western blot, then designed siRNA targeting autophagy related genes (ATGs). When ATGs were inhibited before ppp-siRNA transfection, the efficiency of cancer killing by ppp-siRNA might change a lot. And we can further explain the role of autophagy during the procedure of cancer cell killings.4. Lewis lung cancer cells (LLCs) were injected into C57/BL6mice to build a subcutaneous tumor model. We had25mice and were randomly divided into five groups. When the tumor grew up to about0.5cm, we started to transfect OH-Con, OH-VEGF, ppp-Con, ppp-VEGF into the tumors (once for three days, and were injected for three times), the untreated group were injected with20ul5%glucose. After2days of the third injection, the mice were executed, and tumor tissues were collected. Immunohistochemistry was performed to observe the intratumoral infiltration of CD31positive tubes. HE staining was used to detect the status of cell necrosis. The remaining tissues were used for mRNA extraction, and detected the level of VEGF, immune molecules, apoptosis-and autophagy-related genes. To validate that ppp-VEGF could inhibit tumor angiogenesis, stimulate innate immunity, and induce cancer cell death in vivo.Results1. Anti-angiogenesis effects of ppp-VEGF:Lung cancer cells transfected with OH-VEGF and ppp-VEGF showed lower mRNA level of VEGF compared with OH-Con, and ppp-Con.(OH-Con vs OH-VEGF: P <0.01; ppp-Con vs ppp-VEGF: P<0.05). The VEGF silencing function of OH-VEGF and ppp-VEGF was further validated in protein level by western blot and Elisa (OH-Con vs OH-VEGF:P<0.05; ppp-Con vs ppp-VEGF:P<0.05). We performed scratch assay on HUVECs, and found that HUVECs treated with ppp-VEGF had significantly lower migration rate (OH-Con vs OH-VEGF:P<0.01; ppp-Con vs ppp-VEGF:P<0.01). We then detected the tube formation of HUVECs in Matrigel, and it demonstrated that HUVECs transfected with ppp-VEGF has shorter tube length and fewer branch points (P<0.01).2. ppp-VEGF activated anti-tumor immunity:After transfection with ppp-Con and ppp-VEGF, the expression of IP-10and IFN-β in lung cancer cells was notably increased in mRNA level (OH-Con vs ppp-Con: P <0.01; OH-VEGF vs ppp-VEGF:P<0.01). In the supernatant of A549cell line, the protein expression of the above immune molecules was also elevated a lot. Meanwhile, the amount of MHC-I/HLA positive cells were significantly raised (in A549cells:OH-Con vs ppp-Con:P<0.05; OH-VEGF vs ppp-VEGF:P<0.05; in LLC cells:OH-Con vs ppp-Con:P<0.01; OH-VEGF vs ppp-VEGF:P<0.01). It meant that ppp-siRNA could inhibit the immune evasion of cancer cells. ppp-siRNA could notably increase the expression of RIG-I and the downstream molecule MAVS. When we inhibit RIG-I and MAVS with RNAi technology before ppp-siRNA transfection, the expression of IP-10and IFN-decreased a lot compared with single agent of ppp-siRNA, suggesting that ppp-siRNA induced innate immune system through the RIG-I signaling pathway.3. ppp-VEGF induced endogenous apoptosis and incomplete autophagy:A549cells treated with ppp-siRNA showed higher percentage of Annexin-V positive cells. MTT and trypan blue count demonstrated that the cell viability decreased significantly in ppp-siRNA treated groups (P<0.05). They suggested that ppp-siRNA could induce apoptosis, and inhibit the proliferation and survival of cancer cells. Cancer cells transfected with ppp-siRNA had higher mRNA level and protein level of Noxa, suggesting ppp-siRNA might play the function of cancer cell killings through endogenous apoptosis pathway. To be followed, ppp-siRNA could increase the ratio of LC3-II/LC3-I. If autophagy-related genes were silenced24h before ppp-siRNA treatment, the cell killing rate would decrease notably. It provided us with the theory that ppp-siRNA might kill cancer cells through endogenous apoptosis and incomplete autophagy.4. ppp-VEGF still worked when applicated in vivo:After getting the tumor tissues from the mice. We first detected the mRNA level of VEGF in the tumors, and found that the expression of VEGF would be decreased significantly by OH-VEGF and ppp-VEGF (OH-Con vs OH-VEGF: P <0.05; ppp-Con vs ppp-VEGF: P <0.01). And immunochemistry staining of CD31showed the vascular network formation was also inhibited. Furthermore, the immune molecule IL-12was stimulated by ppp-siRNA (P<0.05). HE staining demonstrated that cell necrosis was much more often found in ppp-siRNA treated tissues. In vivo, ppp-VEGF could inhibit tumor angiogenesis, induce innate immune, and promote cancer cell deaths.Conclusions1. ppp-VEGF could inhibit tumor angiogenesis: in vitro, ppp-VEGF could specific silence VEGF expression, block the proliferation, migration, and tube formation in HUVECs; in vivo, we injected ppp-VEGF into the tumors, and VEGF expression was decreased, the vascular density in tumor could also be reduced.2. ppp-VEGF could induce anti-tumor immunity:ppp-VEGF could activate the expression of IFN-β and a variety of cytokines through RIG-I signaling pathway, resulting in anti-tumor immunity; Meanwhile, ppp-VEGF could up-regulate the expression of MHC-I, inhibit the cancer cell mediated immune evasion.3. ppp-VEGF could promote cancer cell death:ppp-siRNA could stimulate endogenous apoptosis pathway, and induce incomplete autophagy in cancer cells, promoting cancer cell death.4. Multi-functional short interfering RNA (ppp-VEGF) could inhibit tumor angiogenesis, induce innate immune, and promote cancer cell death at the same time. It overcomes the shortcomings of traditional anti-angeiogenic agents:1. Must be combined with chemotherapy;2. Secondary drug resistance. It might provide a theoretical basis for the development of new multi-functional anti-tumor RNAi drugs, and it might have the value for clinical translation and industrialization prospects. |
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